THE JOURNAL
`OF
`
`EXPERIMENTAL MEDICINE
`
`EDITED BY
`MACLYN McCARTY
`RENE DUBOS
`HENRY G. KUNKEL
`
`ADVISORY EDITORS
`
`VINCENT G. ALLFREY
`
`JEROME GROSS
`
`JAMES G. HIRSCH
`ALEXANDER G. BEARN
`FRANK L. HOR SF AL L, JR.
`B AR UJ BENACERRAF
`RU PERT E. BILLINGHAM COUNCILMAN MORGAN
`HANS J. M'ULLER - EBERHARD
`ALBERT H. COONS
`C HANDLER A S T ETSON
`JONATHA N W. UHR
`W . BARRY WOOD, JR.
`
`FRANK J. D I XON
`HARRY EAGLE
`HAROLD S. G I NSBERG
`
`VOLUME 132
`1970
`
`PUBLISHED MONTHLY BY
`
`T HE ROCKEFELLER UNIVERSITY PRESS
`
`LIBRARY
`'UNIVERSITY OF CALIFOR.m4
`DAVIS
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`COPYRIGHT@ 1970 BY THE ROCKEFELLER U NIVERSITY PRF.SS
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`CONTENTS
`
`l O. 1, JULY 1, 1970
`
`1
`
`31
`
`44
`
`PAGE
`G. L. Asn:ERSON and M. ZEMBAI.A . Contact sensitivity in the mouse. IV.
`The role of lymphocytes and macrophages in passive transfer and the
`mechanism of their interaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`J3ARRY R. BLOOM, Lms JIMENEZ, and PHILIP I. lVIARcus. A plaque assay
`for enumerating antigen-sensitive cells in delayed-type hypersen-
`sitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`·
`. . . . . . . . 16
`ERWIN DIENER and MARC .fELDll'IANN. Antibody-mediated suppression of
`the immune response in vitro. II. A new approach to the phe-
`nomenon of immunological tolerance. . . . . . . . . . .
`RooERT C. SEEGER and JoosT J. OPPENHEIM. Synergistic interaction of
`macrophages and lymphocytes in antigen-induced transformation of
`lymphocytes... .
`. . . . . . . . . . . . . . . . . . . . .
`D. C. BENJAMIN and W. 0. WEIGLE. The termination of immunological
`unresponsiveness to bovine serum albumin in rabbits. I. Quantita-
`Live and qualitative response to cross-reacting albumins. . . . . . .
`Bmc;1~R ANDERSSON. Studies on the regulation of avidity at the level of
`the single antibody-forming cell. The effect of antigen close and time
`after immunization . . . . . . . . . . . . .
`. . . . . . . . .
`G101<CIO TONJETTI, MICHAEL B. A. OLDSTONE, and FRANK J. DIXON. The
`effect of induced chronic viral infections on the imm.unologic diseases
`of New Zealand mice . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . .
`H. WIGZELL and O. MA.KELA. Separation of normal and immune lymphoid
`cells by antigen-coated columns. Antigen-binding characteristics of
`membrane antibodies as analyzed by ha.pten- protein antigens. . .
`s. FAI CHEUK, HELMUT H. HAHN, DOUGLAS M . .MOORE, DONALD N .
`KRAUSE, PETER A. TOMASULO, and W. BARRY WOOD, JR. Studies
`on the pathogenesis of fever. XX. Suppression and regeneration of
`pyrogen-producing capacity of exudate granulocytes. . . . . . . . . . . . . . 127
`D. C. GILMOUR, G. A. THEIS, and G. J. TliORBECKE. Transfer of anti-
`body production with cells from bursa of Fabricius .. . ... . .. . . . . . . . 134
`SUSAN ZOLLA, JOEL BuxBAUM, E. C. fnANKLIN, and :iVI. D. SCHARFF.
`Synthesis and assembly of immunoglobulins by malignant human
`plasmacytes. I. Myelomas producing -y-chains and light chains . . . . 148
`]. WAYNE STREILEIN and R . E. BILLINGHAM. An analysis of graft-versus(cid:173)
`bost disease in Syria.n hamsters. I. The epidermolytic syndrome:
`description and studies on its procurement. . ..... . ..... . ... . .. .. 163
`
`66
`
`77
`
`89
`
`110
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`J. WAYNE STEILEIN and R. E. BILLINGHAJvC. An analysis of graft-versus(cid:173)
`host disease in Syrian hamsters. II. The epiderrnolytic syndrome:
`studies on its pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
`
`PAGE
`
`199
`
`No. 2, AUGUST 11 1970
`A. L . L UZZATI, R. j\I[. Tosr, and A. 0. CAR!lONARA. Electrophoretically
`homogeneous ant ibody synthesized by spleen foci of irradiated
`repopulated m ice.... . . . . . .
`. . . . . . . . . . .
`TAX TE \i\fu and ELVIN A. KABAT. An analysis of the sequences of the
`variable regions of Bence J ones proteins and myeloma light chains
`and their implications for antibody complementarity. . . . . . . . . . . . 211
`J AMES H . GORDON and R ENE Dunos. The anaerobic bacterial flora of the
`mouse cecum .. . . .. ... . . .. . . ........ . . . . . .. . ..... .. . . .. . ... . . . 251
`DAVID H. KATZ, Wu.LIAM E. PAUL, EDMOND A. GomL, and BARUJ
`BENACERRAF. Carrier function in anti-hapten immune responses.
`I. Enhancement of primary and secondary anti-hapten antibody
`responses by carrier preimmunization . . . . . . . . . . . . . . . . . . . . . . . . . . 261
`WrLLIAM E. PAUL, DAVID H. K.ATz, EDMOND A. GomL, and BARUJ
`BENACERRAF. Carrier function in anti-hapten immune responses.
`II. Specific properties of carrier cells capable of enhancing anti-
`hapten antibody responses . .. .. ..... ... .. .. . .. . . ... ... .. . ... . . . 283
`ROBERT C. SKARNES. H ost defense against bacterial endotoxernia:
`m.echanism in normal animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
`HENRY R. HitGARJ). Synergism of thymus and bone marrow in the pro(cid:173)
`duction of g raft-versus-host splenomegaly in X -irradiated hosts. .. . . 317
`PETER C. H ARPEL. Human plasma alpha 2-macroglobulin. An inhibitor of
`plasma k allikrein. . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
`J AllillS C. K ENNEDY, P ERRY E . TREADWELL, and EDWIN S. LENNOX.
`Antigen-specific synergism in the immune r esponse of irradiated mice
`given marrow cells and peritoneal cavity cells or extracts ... .... . 353
`PAvlI> OsonA. Some physical and radiobiological properties of immu-
`nologically r eactive mouse spleen cells . . . .
`. . . . . . . . . 368
`
`No. 3, SEPTEMBER 1, 1970
`L. vV. CLEM and P.A. SMALL, J1~. P hylogeny of immunoglobu lin structure
`and function. V. Valences and association constants of teleost anti-
`bodies to a haptenic determinant . . . . . . . . . . . . . . .
`. .... . . . . . . .. 385
`RICHARD J. M ANGl and MICHAEL R. MARDINEY, JR. The in v itro trans(cid:173)
`formation of frozen-stored lymphocytes in the rn.ixed lymphocyte
`reaction and in cul ture with phytohemagglutinin and specific antigens . 401
`MARTINE Y. K. ARMSTRONG, ERNST GLEICIIMANN, HELGA GLEICHMANN,
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`v
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`448
`
`l'AGE
`Lo1mAINE BELDOTTI, JANINE ANm~E-SCHWARTz, and ROBERT S.
`ScHWAll'fZ. Chronic allogeneic chsease. II. Development of lym-
`phomas. . . . . . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
`Nm:r, L. WARNER and LEONORE A. HERZENBERG. Tolerance and im(cid:173)
`munity to maternally derived incompatible IgG2 .. -globulin in mice. . 440
`f.RED G. GuDAT, T. N. HARRIS, SUSANNA HARRIS, and KLAUS HUMMELER.
`Studies on antibody-producing cells. I. Ultrastructure of 19$ and
`7S antibody-producing cells .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`JuDITI-1 C. KNECHT, GERALD SCHIFFMAN, and ROBERT AUSTRIAN. Some
`biological properties of pneumococcus Type 37 and the chemistry of
`its capsular polysaccharide . . . . . .. ...... ...... . ....... . ......... 475
`Dov L. Bonos and KENNETH S. WARREN. Delayed hypersensitivity-type
`grnnuloma formation and dermal reaction induced and elicited by
`a soluble factor isolated from Schistosorn.a mansoni eggs. . . . . . . . .
`H. G. KUNKEL, F. G. JOSLIN, G. M. PENN, and J. B. NATVIG. Genetic
`variants of ')'G4 globulin. A unique relationship to other classes of
`')'G globulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`R. J. NORTH. The relative importance of blood monocytes and fixed
`macrophages to the expression of cell-mediated immunity to infec-
`tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`R. J. NORTH. Suppression of cell-mediated immunity to infection by an
`antimitotic drug. Further evidence that migrant macrophages ex-
`press immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`:Efox. B. RICHERSON, HAROLD F. DvoRAK, and SIDNEY LESKOWITZ.
`Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote
`reaction, general characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`HAlWLD F. DVORAK, ANN M. DVORAK, BLANCHE A. SIMPSON, HAL n.
`RICHERSON, SIDNEY LESKOWITZ, and MORRIS J. KARNOVSKY. Cu(cid:173)
`taneous basophil hypersensitivity. IL A light and electron microscopic
`description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`OsrAs STU'.1'.MAN, EDMOND J. YUNrs, and ROBERT A. GooD. Studies on
`thymus function. L Cooperative effect of thymic function and
`lymphohemopoietic cells in restoration of neonatally thymectomized
`mice.... . . . .. .. ........ .... . .. . .. ..... . . . .
`OSIAS STUT:MAN, EDMOND J. YUNIS, and ROBERT A. Goon. Studies on
`thymus function. IL Cooperative effect of newborn and embryonic
`hemopoietic liver cells with thymus function .. . . .... .. . . . . . ..... 601
`
`488
`
`508
`
`521
`
`535
`
`546
`
`558
`
`583
`
`No. 4, OCTOBER 1, 1970
`EDNA MOZES, G. M. SHEARER, and MICHAEL SELA. Cellular basis of the
`genetic control of immune responses to synthetic polypeptides. I.
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`l'AG£
`
`613
`
`623
`
`694
`
`702
`
`721
`
`Differences in frequency of splenic precursor cells specific for a syn(cid:173)
`thetic polypeptide derived from multichain polyproline ([T, Gj-
`Pro- -L) in high and low responder inbred mouse strains.
`G. CunKow1cz, G. M. SHEARER, and T. ho. Cellular differentiation
`of the immune system of mice. VI. Strain differences in class dif-
`ferentiation and other properties of marrow cells. .
`J. I). DunEY, NANCY L. MILLER, and ]. IC FRENKEL. The Towplasnza
`gondii oocysl from cat feces. . . . . .
`. . . . . . . . . . . . . . . . . . . . . . . . . . . 636
`STEPHEN I. MORSE and BRUCE A. BARRON. Studies on the leukocytosis
`and lymphocytosis induced by Bordetelta pertitSSt-s. III. The distribu-
`. tion of transfused lymphocytes in pertussis-treated and normal
`mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663
`Fl~ANK R. ScmnD, IVAN M. RoITT, and l\IIA.1uA J. ROCHA. Complement
`fixation by a
`two-component antibody system : immunoglobulin
`G and immunoglobulin lVI anti-globulin (rheumatoid factor). Para-
`doxical effect related to immunoglobulin G concentration . .... . . . .. 673
`GARY D. OVERTURF and EDWARD A. MORTIMER, ]R. Studies on the
`relationship between the production of bacteriocines by Group A
`streptococci and acute glomerulonephritis.... . ...... . .........
`CELSO IlrANCo, RICHARD PATRICK, and VICTOR NussENZWEIC. A popula(cid:173)
`tion of lymphocytes bearing a membrane receptor for antigen(cid:173)
`antibody- complement complexes. I. Separation and characteriza-
`tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`ALAN E . BEER and R. E. BILLINGHAM.
`Implantation, transplantation,
`and epithelial-mesenchymal relationships in the rat uterus. . . . . . .
`MICHAEL PorrnR and RosE LIEBERMAN. Common individual antigenic
`determinants in five of eight BALB/c IgA rnyeloma proteins that
`bind phosphoryl choline . ... . . . . . . . . . . . . . . . . . . . . .
`. . . 737
`Gurno Bioz:a, RICHARD AsOFSKY, RosE LIEBERMAN, CLAUDE STIF.li'EL,
`DENISE Mou'.I'ON, and BARUJ B ENACERRAF. Serum concentrations
`and allotypes of immunoglobulins in two lines of mice genetically
`selected for "high" or "low" antibody synthesis...... ... .......
`B . SHANNON DANES, J. E. SCOTT, and ALEXANDER G. BEARN. Further
`studies on metachromasia in cultured human fibroblasts. Staining
`of glycosaminoglycans (mucopolysaccharides) by Alcian b lue in salt
`solutions . . . .. ...... .. . . . .. .. . ..... . . .... . ...... . . . ...... . .. . . 765
`NEIL R. COOPER and HANS J. MuLLER-ElrERHARD. The reaction
`mechanism of human CS in immune hemolysis . . ..... . ........ . . 775
`RAJ.PH VAN FURTH, ]AMES G. HmscH, and MARTHA E. F.rmoRKO. Mor(cid:173)
`phology and peroxidase cytochemistry of mouse promonocytes,
`rnonocytes, and macrophages.
`. . . . . . . . .
`
`752
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`794
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`Vil
`
`l'AGE
`
`RALPH VAN F URTH and MARTINA .M. C. DmssELHOFF-DEN DULK. The
`kinetics of promonocytes and monocytes in the bone marrow. . . . . . 813
`D. SJ·:J.LlN, M . Ki:ivARY, U. ROTHER, and K. ROTHER. Intrarenal com-
`plement fixation by cytotoxic antibodies . . . . . . . . . . . . . . . . . . . .
`
`829
`
`' 1
`
`858
`
`868
`
`885
`
`898
`
`916
`
`No. 5, NovmmER 1, 1970
`v. s. HYERS and E. E. SERCARZ.
`Induction and reversal of immune
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
`paralysis in vitro.. . . . . . .
`M. VAN WrNKLl': and L. LEVY. Further studies on the reversibility of
`serum sickness cholesterol-induced atherosclerosis. . . . . . . . . .
`PETER B. LAMBERT and HOWARD A. FRANK. Cellular and vascular com(cid:173)
`ponents of
`the allograft reaction. Evidence from returned skin
`nllografts...
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`NATHAN TRAININ and MYRA SMALL. Studies on some physicochemical
`properties of a thymus humeral factor conferring immunocom-
`petence on lymphoid cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`OTTO GOTZE and HANS J. ::V.l"tiLLER-Enm:rnARD. Lysis of erythrocytes
`by complement in the absence of antibody. . . . . . . . . . .
`PE'f8R J. McCuLLAGH. The abrogation of sheep erythrocyte tolerance
`in rats by means of the transfer of allogeneic lymphocytes.. . . . . .
`WESLEY \V . BULLOCK and MARVIN B. RITTENBERG . In vitro-initiated
`secondary anti-hapten response. II. I ncreasing cell avidity for
`antigen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926
`N. K. B. DAY, H. GEwunz, R . JOHANNSEN, J. FINSTAD, and R. A.
`Goon. Complement and complement-like activity in lower ver(cid:173)
`tebrates and invertebrates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941
`BRUCE W. B1UENT and ALFRED NrsoNOFF. Quantitative investigations of
`icliotypic antibodies. IV. Inhibition by specific haptens of the reaction
`of anti-hapten antibody with its anti-idiotypic antibody.. . . ........ 951
`H. F. JEEJEEBHOY. T he effect of heterologous anti-lymphocyte serum
`on lymphocytes of thymus and marrow origin. . . . . . . . . . . . . . . . . . . . 963
`KNUD D. KNUDSEN, LEWIS K . DAHL, I(EITH T HOMPSON, J UNICHI lWAI,
`MARTHA HEINE, and GEO.RGE LEITL. Effects of chronic excess salt
`ingestion. Inheritance of hypertension in the rat. . . . . . . .
`BENVENUTO PERNIS, LUCIANA FORNI, and L UISA AMANTE. Immuno(cid:173)
`globulin spots on the surface of rabbit lymphocytes . . . . . . . . . . . . . . . . 1001
`VOLI<l·:tl SCHIRRMACHER and KLAUS RAJEWSKY. Determination of anti-
`body class in a system of cooperating antigenic determinants. . . . . . 1019
`R. V. 8r,ANDEN. Mechanisms of recovery from a generalized viral infec-
`tion : mousepox. I. The effects of anti-thyrnocyte serum. . . . . . . . .
`
`976
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`1035
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`PAGE
`
`No. 6, DECEMBER 1, 1970
`IRUN R. COHEN, LARY STAVY, and MICHAEL FELDMAN. Glucocorti(cid:173)
`coids and cellular immunity in vitro. Facilitation of the sensitization
`phase and inhibition of the effector phase of a lymphocyte anti-
`fibroblast reaction ...... .. ... . ... . .. . .. .... ................... 1055
`ELLEN BoRENFREUND, YuJI HoNoA, Mn.DRED STEINGLASS, and AARON
`BENDICH. Studies of DNA-induced heritable alteration of mam-
`malian cells .... .. ... ...... . ....................... .. ..... . .. 1071
`JOSEPH D. SCHULMAN and KATIIl<YN H. BRADr;J·:Y. The metabolism of
`amino acids, peptides, and clisulfides in lysosomes of fibroblasts cul-
`tured from normal individuals and those wit11 cystinosis .. ...... . . .. 1090
`MICHAEL HEIDELBERGER and MOREY E. SLODKC. Predicted and un(cid:173)
`predicted cross-reactions of an acetylphosphogalactan of Sporobolo-
`myces yeast. II . ... .... ................... . ..... ... ......... .. 1105
`D. G. COLLEY, A. Y. SmH Wu, and B. H . WAKSMAN. Cellular differenti-
`ation in the thymus. III. Surface properties of rat thymus and lymph
`node cells separated on density gradients .............. .......... 1107
`HAROLD C. MILLER and GusTAvo Cunxow1cz. Antigen-specific cells in
`mouse bone marrow. I. Lasting effects of priming on inununocyte
`production by transferred marrow .............................. 1122
`JOEL G. BASEMAN, A. M. PAPPEN'HEThfER, Jn., D. M. GILL, and ANNABEL
`A. HARPER. Action of diphtheria toxin in the guinea pig.....
`M. A. VEJ\KATACHALAM, M. J. KARNOVSKY, H. D. FAIUMI, and R. S.
`CoTRAN. An ultrastructural study of glomernlar permeability using
`catalase and peroxidase as tracer proteins. . . . . . . . . . . . . . . . . . . . . . . 1153
`M. A. VENKATACHALAM", R. S. Con<AN, and M. J. KARNOVSKY. An
`ultrastructural study of glomerular permeabili ty in aminonucleoside
`nephrosis using catalase as a tracer protein. . . . . . . . . . . . . . . . . . . . . 1168
`T . TAKAHASm, E. A. CARSWELL, and G. J. TIIORDECKE. Surface anti-
`gens of im1mmocompetent cells. I. Effect of 0 and PC.l alloantisera
`on the ability of spleen cells to transfer immune responses. . . . . . . . . . 1181
`N. ABRAMSON, A. F. LoBucuo, J. H. J ANDL, and R. S. CoTRAN. The
`interaction between human monocytes and red cells. Binding char-
`acteristics...................... ...... ................. .
`N. ABRAMSON, E. W. GELFAND, J. H. JANDL, and F. S. RosEN. The in(cid:173)
`teraction between human monocytes and red cells. Specificity for JgG
`subclasses and IgG fragments .................................. 1207
`J. J. T. OWEN and M. C. RAFF. Studies on the differentiation of thymus-
`derived lymphocytes ..... . .. . .......................... ....... 1216
`CURLA S. WALTERS and HA:-.1S WIGZELL. Demonstration of heavy and
`light chain antigenic determinants on the cell-bound receptor for
`
`1138
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`1191
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`l'AGE
`
`anligen. Similarities between membrane-attached and humoral
`antibodies produced by the same cell. . . . . . . . . . . . . . . . . . . . . . . . . 1233
`CHAJU,ES HUGGINS, SOL WISEMAN, and A. H. REDDI.
`'11-ansformation
`of fibroblasts by allogeneic and xenogeneic transplants of deminera-
`lizecl tooth and bone. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1250
`vVII.LlAM JOHN MARTIN", LEONARD ELLMAN, IRA GREEN, and BARUJ
`:BENACERRAF. Histocompatibility type and immune responsive-
`ness in random bred Hartley strain guinea pigs. . . . . . . . . . . . . . . . . . 1259
`KLAUS-ULRICH HARTMANN.
`Induction of a hemolysin response in vitro.
`Interaction of cells of bone marrow origin and thymic origin.. ... . . 1267
`JEJ\N-CLAUDE BYSTRYN, MARTIN W. GRAF, and JONATHAN W. UHR.
`Regulation of antibody formation by serum antibody. II. Removal
`of specific antibody by means of exchange transfusion . . . . . . .
`
`1279
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`THE JOURNAL OF EXPERIMENTAL MEDICINE
`JULY 1, 1970
`VOLUME 132, NUMBER 1
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`AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS
`OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS
`AND THEIR IMPLICATIONS FOR ANTI-
`BODY COMPLEMENTARITY*
`
`BY TAI TE WU, PH.D., AND ELVIN A. KABAT, PH.D
`(From the Departmetits of Microbiology, Neurology, and H1tmati Genetics and Develop(cid:173)
`mem, College of Physicia11,s and Surgems, Columbia Utiiversity, and the Ne1iro(cid:173)
`logical fostitute, Presbyteriati Hospital, New York 10032; the Biomathematics
`Divisioti, Grad1ro.te School of Medical Sciemes, Comell University and the
`Sloan-Kettering Instiifite, New York 10021)
`
`(Received for publication 26 March 1970)
`
`The extraordinary versatility of the antibody-forming mechanism in producing an
`almost limitless number of specific receptor sites complementary for almost any molec(cid:173)
`ular conformation of matter within a size range (1-3) represented by a hexa- or hepta(cid:173)
`saccharide as an upper and a mono- or disaccharide as a lower limit, is almost certainly
`related to the unique structural features of immunoglobulins and difierentiates them
`from all other known proteins. These antibody-combining sites are formed as a con(cid:173)
`sequence of the interaction of two polypeptide chains, a light and a heavy chain (2, 4,
`5). The antibodies usually formed to various antigens often represent heterogeneous
`populations of immunoglobulin molecules of different classes, subclasses, and genetic
`variants and also show specificities t~ward different antigenic determinants (1, 2, 6, 7) .
`In some instances, however, relatively homogeneous populations of antibodies with
`respect to many of these properties have been obtained. Among these have been human
`antibodies to dextran and levan (8, 9) and rabbit antibodies to the group-specific carbo(cid:173)
`hydrate of streptococcus (10- 12), antibodies to the Type III-specific capsular poly(cid:173)
`saccharide of pneumococcus (13, 14), rabbit antihapten (15), and specimens of anti(cid:173)
`bodies and of Fab' fragments which crystallized (Nisonoff et al., in references 16, 17),
`but sequence data on these are not yet available.
`The large body of sequence data related to immunoglobulin structure comes from
`the analysis of urinary Bence Jones proteins and from the monoclonal immunoglobu(cid:173)
`lins found in large amounts in the sera of patients with multiple myeloma and Walden(cid:173)
`strom macroglobulinemia (16, 18). While a substantial body of evidence was available
`relating these proteins to immunoglobulins, the recent demonstration that many
`myeloma globulins have specific ligand-binding properties like those of many anti(cid:173)
`bodies provides increasing confidence that myeloma globulins represent homogeneous
`populations of antibody molecules (16, 18-27). The ability to produce in BALB/c
`
`* Aided by grants from the National Science Foundation (GB-8341) and the National
`Cancer Institute (CA-08748), and a general Research Support Grant of the U. S. Public
`Health Service.
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`.BENCE JONES PROTEINS AND MYELOMA LIGFr1' CHAINS
`
`mice myelomas and macroglobulinemias (28) which produce myeloma globulins and
`Bence Jones proteins like those in the human, provides a source of data Crom which
`important evolutionary trends can be inferred.
`T hus the extensive sequence data on Bence Jones proteins, which are considered to
`be light chains of myeloma globulins and Waldenstrom macroglobulins (29), and on
`various light and heavy chains, provide information clearly pertinent to the problem
`of the elucidation of the structure of antibody-combining sites.
`The unique finding that distinguishes the immunoglobulins from all other proteins is
`that the N-terminal half of the light chains and the N-terminal quarter of the heavy
`chains vary in sequence in samples obtained from individual monoclonal immuno(cid:173)
`globulins and that indeed no two such variable regions of any chain and no two mye(cid:173)
`loma immunoglobulins or Bence Jones proteins have thus far been found to be identical
`in sequence (30). The constant region, however, is essentially no different from other
`proteins in that the variation in the amino acidc; found at any position is ascribable
`to species and class variations or to genetic variants such as Inv factors. By com(cid:173)
`parison of sequence data on the variable and constant regions of Bence Jones proteins
`with amino acid composition of purified human antibodies, it could be shown that
`most of the compositional variation could only originate in the variable region (see
`Kabat in reference 18).
`From sequence data, a variety of hypotheses have been advanced (7, 31-35) to
`explain the structural basis of antibody complementarity. All of these are selective
`theories, i.e. they consider that the information for complementarity is essentially built
`into the primary sequence of each chain and that a given antigen only triggers the
`biosynthesis of those antibody molecules having complementary receptor sites. There
`are two types of selective theories: germ line theories (36) and somatic mutation
`theories (37-39). At present no hypothesis is generally accepted. Excellent reviews
`(sec above) arc available.
`
`The present communication is an extension of earlier efforts from this labora(cid:173)
`tory (18, p. 87, and 40-43) to locate more precisely those portions of the vari(cid:173)
`able region which are directly responsible for antibody complementarity, that
`is which make direct contact with the antigenic determinant, and to explain
`the unique capacity of these proteins to have so many complementary regions.
`As in the earlier studies, all human K, human X, and mouse K .Bence Jones
`protein and light chain sequences are aligned for maximum homology (44)
`and all variable regions are considered as a unit and compared with the con(cid:173)
`stant regions. These earlier studies had called attention to the following:
`(a) The variable regions had fe:w if any species-specific positions while the
`constant regions of the human and mouse proteins had 36 species-specific
`amino acid substitutions per 107 residues (40, 45). A species-specific position
`is defined as one at which the amino acid residues in the mouse proteins differ
`from tl1ose in the human proteins.
`(b) When the invariant residues of these two regions were compared, the
`latest tabulation (45) showed the variable regions to have 10 invariant and
`almost invarianl glycines and no invariant alanines, leucines, valines, histi-
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`TAI TE WU AND ELVIN A. KABAT
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`213
`
`dines, lysines, or serines while lhe constant regions had 3 each of invariant
`alanine, leucine, and valine, and 2 invariant histidines, 2 invariant lysines, and
`5 invariant serines. It was suggested that the invariant glycines were important
`in contributing to the flexibility needed by the variable region in accommo(cid:173)
`dating the numerous substitutions (41, 43) at the variable positions. It was
`also suggested that the invariant glycines near the end of the variable region
`at positions 99 and 101, plus the almost invariant glycine at position 100,
`provided a pivot upon which the complementarity-determining regions might
`move to make better contact with the antigenic determinant (43; 18, p. 87)
`just as the walls of the lysozyme site have been shown to adjust somewhat to
`accommodate its hexasaccharide substrate (46). The hydrophobic residues in
`the constant region were hypothesized to be involved in noncovalent bonding
`to the heavy chain.
`(c) From an examination of sequences of the KI, KII, and K!II subgroups
`(Hood et al. in reference 16) (47, 48) of the human Bence Jones proteins in
`which many of the proteins in a subgroup had an identical sequence for the
`first 20- 24 residues, it was postulated that there are two kinds of residues in
`the variable regions, those making direct contact with the antigenic determi(cid:173)
`nant (complementarity determining) and those which are involved only in
`three-dimensional folding (42). The latter would be expected to have less
`stringent requirements, and more mutation noise would be permitted than
`with the complementarity-determining residues. This distinction led to the
`inspection of the sequences for short stretches showing very high variability
`and two of these were identified: the most variable beginning at residue 89
`and ending at 97, the other running from residue 24 through 34. Each of these
`two unusually highly variable regions began after an invariant half-cystine
`and was followed by an invariant phenylalanine (residue 98) and an invariant
`tryptophane (residue 35) respectively. It is of interest thal the two regions are
`brought close together by the S-S bond I2:rlI88 (45). Milstein (47), Milstein
`and Pink (7), and Franek (49) have also called attention to the highly variable
`positions in these regions and Franek (49) has noted an additional highly
`variable region around residues 52-55. It was hypothesized (45) that these first
`two regions might represent the complementarity-determining regions and
`that complementarity might be acquired by the insertion of small linear se(cid:173)
`quences into the light and heavy chains by some episomal or other insertion
`mechanism. It is striking that the differences in chain length seen in the Bence
`Jones proteins ar~ confined to these two regions of the chain. T he remaining
`portions of each chain would be essentially under the control of structural
`genes. The inserted sequences would be drawn from a large but finite set and
`either inserted under the influence of antigen, if antibody-forming cells are
`multipotent, or individual sequences might be distributed to immunoglobulin-
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`214
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`BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS
`
`formfog cells during differentiation iI t he capacity of individual cells to synthe(cid:173)
`size antibody is restricted.
`This working hypothesis offers several advantages:
`(a) It is capable of providing the evolutionary stability and accounts for
`the universality of the antibody-forming mechanism throughout the verte(cid:173)
`brates. Germ line theories (34- 36) postulate one gene for each of the thousand
`or more variable regions (30). This would be expected to result in divergence
`during evolution since the loss by mutation of any one variable region would
`only minimally affect the capacity to form antibody and survival; thus indi(cid:173)
`viduals and populations lacking certain variable regions would arise.
`(b) It offers a substantial simplification to the problem of producing a very
`large number of complementary sites. While it is known that in all proteins
`with specific receptors the site is formed by residues from widely separated
`portions of the chain, these sites are all formed by single chains. Thus, form(cid:173)
`ing a three-dimensional site must involve residues from various regions. The
`antibody site being formed by a heavy and a light chain need not necessarily
`be so restricted.
`Since much additional data on the light chains and a number of heavy chain
`sequences have been accumulated, the present communication represents a
`further attempt at analyzing the unique features of the variable regions of
`immunoglobulin chains. Among aspects considered are the role of glycine,
`invariant residues, and hydrophobicity patterns, and the highly variable por(cid:173)
`tions, with a view to localizing the regions responsible for complementarity
`and evaluating various theories in terms of evolutionary origin and perpetua(cid:173)
`tion of the antibody-forming mechanism.
`Sequence Data Employed-Complete and partial sequence data have been
`published on 77 Bence Jones proteins and immunoglobulin light chains as well
`as on a number of heavy chains. Data were available on 24 human KI, 4 human
`K!I, 17 human KlII, 10 human >..I, 2 human >..II, 6 human >..III, 5 human >..IV,
`2 human ;\ V, 2 mouse Kl, and 5 mouse Kil proteins. 1
`
`The original light chain sequence data may be found in the following references.
`HBJ 98: Baglioni, C. 1967. Bioc/re;m. Biophys. Res. Commun. 26:82.
`Eu: Cunningham, B. A., P. D. Gottlieb, W. H. Konigsberg, and G. M. Edelman. 1968.
`Biochemistry. 7:1983.
`Mil (human Kil): Dreyer, W. J., W. R. Gray, and L . Hood. 1967. Cold Spring Harbor
`Symp. Qtiant. Biol. 32:353.
`Hae, Dob, Pal: Grant, A., and L. Hood. Unpublished work.
`Roy, Cum: Hilschman, N., and L. C. Craig. 1965. Proc. Nat A cad. Sci. U.S. A. 63:1403;
`Hilschmann, N. 1967. Hoppe-Seyler's z. Pltysiol. Chem. 348:1077; Hilschmann, N.,
`H. U. Barnikol, M. Hess, B. Langer, H. Ponstingl, M. Steinmetz-Kayne, L. Suter, and
`S. Watanabe. 1968. Fed. Ettr. Biochem. Soc. Symp., 5t!t. In press.
`
`1 The World Health Organization has recently changed the notation of subgroups so that
`human Kil in this paper will become human ~Ill and human K!II will become human KIL
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`TAl TE WU AND ELVIN A. I<ABAl'
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`215
`
`HS 78, HS 92, HS 94, HS 68, HS 70, HS 77, HS 86, HS 24: Hood, L., and D. Ein. 1968.
`Nature (Londo1i) . 220:764.
`HBJ 7, HBJ 11, HBJ 2, HBJ 8: Hood, L., W. R. Gray, and W. ]. Dreyer. 1966. J . Mo/.
`Biol. 22:179.
`MBJ 41, MBJ 70, MBJ 6: Hood, L., W. R. Gray, and W. ]. Dreyer. 1966. Proc. Nat'l
`Acad. Sci. U.S. A. 66:826.
`HBJ 10, HBJ 1, HBJ 4, HBJ 6, HBJ 5